INTERNATIONAL JOURNAL OF ONCOLOGY 47: 573-582, 2015

Sulphur alters NFκB-p300 cross-talk in favour of p53-p300

to induce apoptosis in non-small cell lung carcinoma
Shilpi Saha1*, Pushpak Bhattacharjee1*, Deblina Guha1, Kirti Kajal1, Poulami Khan1,
Sreeparna Chakraborty1, Shravanti Mukherjee1, Shrutarshi Paul1, Rajkumar Manchanda2,
Anil Khurana2, Debadatta Nayak2, Rathin Chakrabarty3, Gaurisankar Sa1 and Tanya Das1

1
Division of Molecular Medicine, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata-700054;
2
Central Council for Research in Homeopathy, 61-65 Institutional Area, Janakpuri, New Delhi-110058;
3
Bholanath Chakrabarty Trust, 5 Subol Koley Lane, Howrah 711101, India

Received March 25, 2015; Accepted May 25, 2015

DOI: 10.3892/ijo.2015.3061

Abstract. Adverse side effects of chemotherapy during cancer
treatment have shifted considerable focus towards therapies
that are not only targeted but are also devoid of toxic side
effects. We evaluated the antitumorigenic activity of sulphur,
and delineated the molecular mechanisms underlying sulphur-
induced apoptosis in non-small cell lung carcinoma (NSCLC)
cells. A search for the underlying mechanism revealed that the
choice between the two cellular processes, NFκ Bp65-mediated
survival and p53-mediated apoptosis, was decided by the
competition for a limited pool of transcriptional coactivator
protein p300 in NSCLC cells. In contrast, sulphur inhibited
otherwise upregulated survival signaling in NSCLC cells by
perturbing the nuclear translocation of p65NFκ B, its associa-
tion with p300 histone acetylase, and subsequent transcription
of Bcl-2. Under such anti-survival condition, induction of
p53-p300 cross-talk enhanced the transcriptional activity
of p53 and intrinsic mitochondrial death cascade. Overall,
the findings of this preclinical study clearly delineated the
molecular mechanism underlying the apoptogenic effect of
the non-toxic homeopathic remedy, sulphur, in NSCLC cells.
Introduction
In the present scenario lung cancer is the leading cause of
cancer-related deaths worldwide and the incidence of lung
cancer has almost reached epidemic proportions in both devel-
oping and developed countries (1). Despite decades of research,
Correspondence to: Professor Tanya Das or Professor Gaurisankar Sa,
Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme
VII M, Kolkata-700 054, India
E-mail: tanya@jcbose.ac.in
E-mail: gauri@jcbose.ac.in
*
Contributed equally
Key words: apoptosis, Bax, Bcl-2, cancer, homeopathy, NFκB, p300,
p53, sulphur
the available treatment options for lung cancer patients remain
inadequate, either to offer a cure or even a substantial survival
advantage owing to its inherent resistance to chemotherapy.
Moreover, the clinical efficacy and usefulness of chemotherapy
is still limited because of its dose-limiting toxicity (2) which
has considerably shifted the focus towards complementary and
alternative medicine (CAM), that are low in toxic side effects
(3). Among different CAM regimens, homeopathy a nearly
200-year-old system of medicine has been shown to decrease
side effects of chemotherapy in cancer patients and possess
antitumorigenic property (4-8). Homeopaths have described
observations that tumors recede from the use of homeopathic
treatment and have, from time to time, documented long-term
recoveries from cancer in response to homeopathic treatment
(4-6). Unfortunately, scientific studies corroborating these
clinical observations are very few. There are only few reports
on the mechanism of action of homeopathic drugs in experi-
mental cancers and cell cultures (9-14).
In the present study, we investigated the basic molecular
mechanism of antitumorigenic effect of sulphur, a promising
homeopathic remedy, on non-small cell lung cancer cells.
Sulphur, the most ancient archetype in the history of our planet,
is used by homeopaths for treating inflammation and cancer
and also in treating other skin diseases (15-18). Potent chemo-
preventive effects have been demonstrated in various in vivo
and in vitro models for sulphur-containing compounds found
in naturally occurring products, such as, onions and garlic
(19,20). Protective effect of sulphur has also been reported
against cytotoxicity in neuroblastoma cells (21). In vitro
treatment with sulphur significantly increased apoptosis in
neuroblastoma cells (22). Reports have stated growth inhibi-
tory and apoptosis-related effects of sulphur on immortalized
human oral keratinocytes and on oral cancer cells (23). Taken
together, these findings indicate a promising anticancer poten-
tial of sulphur. However, the underlying mode of action for its
professed antitumorigenic effect in highly resistant non‑small
cell lung carcinoma (NSCLC) is still unidentified and requires
further study. To the best of our knowledge, therefore, this is
the first report delineating the detailed mechanism of sulphur
on NSCLC cells.
574 Saha et al: Sulphur induces apoptosis in non-small cell carcinoma cells
It is well established that development and growth of
tumor cells are controlled by complex signalling pathways
involved in the regulation of cell death, survival and prolif-
eration. In mammalian cells, the regulatory contribution
of NFκ B and p53 to cancer development and progression
is well documented where inactivation of p53 and hyper-
activation of NFκ B are the common occurrences (24-27). It
has been acknowledged that NFκ B pathway activation renders
inherent resistance to chemotherapy to NSCLC cells appar-
ently via induction of survival and anti-apoptotic proteins
(25,28). Therefore, targeting NFκ B pathway may serve as a
novel approach to regress NSCLC cells. Conversely, tumor
suppressor p53, the ‘guardian of genome’ translates stress
signals into cell cycle arrest or apoptosis, depending on the
balance between pro-apoptotic and anti-proliferative genes
(29-31). Thus, drugs reviving tumor suppressor functions of
p53 will be proficient for targeted cancer therapy. Considering
the deregulation of NFκ B and p53 pathways in numerous
cancers, including NSCLC cells, it is not surprising that exten-
sive cross-talk between these pathways exists at various levels.
In fact, NFκ B activation was shown to play a role in neoplastic
transformation by inhibiting p53 gene expression (32,33).
Moreover, reports have shown that NFκ B by inducing the E3
ubiquitin ligase MDM2 attenuated p53 protein stability (34).
Furthermore, the NFκ B gene promoter is activated by p53
mutants, and p52 subunit of NFκ B can modulate the promoter
activity of p53 target genes (34). Both NFκ B and p53 compete
for co-activators, for example, the histone acetyltransferases
p300 and CBP (35,36). An ideal therapeutic approach should,
therefore, involve tailoring NFκ B-governed survival pathway
in favor of p53-regulated apoptotic pathway to regress other-
wise drug‑resistant NSCLC cells.
The present study investigated the molecular mechanism
underlying the antitumorigenic potential of homeopathic
remedy sulphur, commonly known as a healing mineral. Our
findings revealed that sulphur preferentially induces apoptosis
in NSCLC cells sparing normal cells. Our exploration for the
detailed molecular mechanism revealed that sulphur inhibits
NFκ B-induced Bcl-2 mediated survival pathway while trig-
gering p53-induced Bax mediated apoptosis in NSCLC cells.
In NSCLC cells, the constitutively active NFκ B associates
with p300 and this NFκ B-p300 complex binds to the promoter
region of Bcl-2 thereby leading to transcriptional upregulation
of Bcl2 that in turn endorses activation of survival pathway.
On the contrary, upon sulphur treatment, pro-apoptotic gene
p53 gets activated and occupies p300 to form p53-p300
complex. NF κ B-p300 complex formation, therefore, gets
hampered and the newly formed p53-p300 complex binds to
the promoter region of p53 target gene, Bax thereby leading
to the transcriptional upregulation of Bax that consecutively
directs activation of apoptotic pathway. Sulphur thus plays an
essential role in dictating pro and anti-apoptotic permutation
to create an environment conducive for induction of apoptosis
in NSCLC cells.
Materials and methods
Cell culture. Human non-small cell carcinoma cell line,
A549 was obtained from NCCS, India. Peripheral blood
collected from healthy human volunteers with informed
consent (Institutional Review Board 1382) was centrifuged
over Ficoll-Hypaque density gradient (Amersham Pharmacia,
Uppsala, Sweden) to obtain total peripheral blood mono-
nuclear cells. Cells were routinely maintained in DMEM
supplemented with 10% heat inactivated fetal bovine serum
(Lonza, NH, USA), L-glutamine (2 mM), sodium pyruvate
(100 µg/ml), non‑essential amino acids (100 µM), streptomycin
(100 µg/ml), penicillin (50 U/ml; Invitogen, CA, USA) at 37˚C
in a humidified 5% CO2 incubator. Cells were maintained in
an exponential growth phase for all experiments. Viable cell
numbers were determined by trypan-blue exclusion test.
Treatment of cells. Placebo and sulphur 6C, 30C or 200C were
procured from Hahnemann Publishing Co., India. Cells were
treated with sulphur/placebo of potencies 6, 30 or 200C expo-
sure at the different concentration (10, 15, 20 and 30 µl/ml)
for different time-points (6, 12, 24, 36 and 48 h) to select the
optimum time required for cell killing. To understand the
sequence of events leading to apoptosis, cancer cells were
treated with mitochondrial pore inhibitor CsA (25 µM; Merck,
Germany) for 1 h prior to incubation with sulphur.
Flow cytometry. For the determination of cell death, cells
were stained with 7AAD and Annexin V-FITC and analyzed
on flow cytometry (FACS Verse, BD Biosciences). Electronic
compensation of the instrument was done to exclude overlap-
ping of the emission spectra. A total of 10,000 events were
acquired for analysis using CellQuest software. For the assess-
ment of mitochondrial transmembrane potential, cells were
stained with potentially-sensitive dye Dihexyloxacarbonicao
cyanine (DiOC6, Merck, Germany) during the last 30 min
of treatment at 37˚C in the dark. Fluorescence of retained
DiOC6 was determined flow cytometrically using logarithmic
amplification by CellQuest software (BD Biosciences) (31).
Fluorescence imaging. Chromatin condensation and nuclear
frag­mentation was analyzed using the standard protocol. Briefly,
cells were grown on coverslips, fixed with 3% p-formaldehyde
for 10 min and then permeabilized with 0.1% Triton X-100 for
5 min. Cells were then incubated with 4'6-diamidino-2-phenyl-
indole (DAPI; BD Pharmingen, CA, USA). The morphology of
the cell nuclei was visualized using a fluorescence microscope
(Leitz microscope fitted with epifluorescence illumina­tor
through a 60X aperture oil immersion lens, Carl Zeiss,
Germany). For fluorescence imaging, cells growing on a cover
slip were fixed with 3% p-formaldehyde and were stained with
anti-p53 and anti-p65NFκ B antibodies (SantaCruz, CA, USA),
after permeabilization with Triton X-100, followed by FITC
and TRITC conjugated secondary antibodies, respectively and
visualized with confocal microscope (Carl Zeiss, Germany)
(13,37).
Plasmids, siRNA and transfections. The expression constructs
pcDNA3.0/HA-tagged Iκ Bα-32A/36A [Iκ Bα super-repressor
(Iκ Bα-SR), a kind gift from Dr J. Didonato, The Cleveland
Clinic Foundation], pcDNA3.1-p65NFκ B/p53/Bcl-2 overex-
pression plasmids (2 µg/million cells) were introduced into
exponentially growing cancer cells using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA) according to the protocol
provided by the manufacturer. In a similar manner, p53
 INTERNATIONAL JOURNAL OF ONCOLOGY 47: 573-582, 2015
expression was knocked down in A549 cells using p53-shRNA
(Santa Cruz) and Lipofectamine 2000 (Invitrogen). Stably
expressing clones were isolated by limiting dilution and
selected with G418 sulphate (Cellgro) at a concentration of
400 µg/ml and cells surviving this treatment were cloned and
screened by western blot analysis with specific antibody. A549
cells were transfected with 300 pmol of Bcl-2-/Bax-/control-
ds-siRNA (Santa Cruz) and Lipofectamine 2000 separately
for 12 h. The levels of respective proteins were estimated by
western blotting (37).
Western blotting. To obtain whole cell lysates, cells were
homogenized in lysis buffer (20 mM Hepes, pH 7.5, 10 mM
KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA and
1 mM DTT) supplemented with protease and phosphatase
inhibitor cocktails. Mitochondrial and cytosolic fractions were
prepared according to Lahiry et al (31). For direct western
blot analysis, a total of 50 µg of protein was resolved using
SDS-PAGE and transferred to nitrocellulose membrane for
western blotting using required antibodies e.g., anti-caspase‑9,
anti-caspase‑3, anti-p53 (DO-1), anti-p65NFκ B, anti-Iκ Bα,
anti-Bcl-2, anti-Bax (N-20), anti-cytochrome c and anti-p300.
Thereafter proteins of interest were visualized by chemilu-
miniscence. Equivalent protein loading in cytosolic, nuclear
and mitochondrial fractions were verified using anti-α-actin/
histone H1/MnSOD antibodies (Santa Cruz, CA, USA) respec-
tively.
Co-immunoprecipitation. For the determination of direct inter-
action between two proteins, co-immunoprecipitation technique
was employed (38,39). p53-p300 and p65NFκ B-p300 interac-
tion was determined by co-immunoprecipitation. Samples
(300 µg of protein from the total lysate) were incubated at 4˚C
overnight with anti-p300/-IgG antibody and then incubated
for 2 h at 4˚C with protein A-Sepharose. Immunocomplexes
were washed of unbound proteins with cold TBS with protease
inhibitors, and pelleted beads were boiled for 5 min in
SDS-PAGE sample buffer. The immunoprecipitated proteins
were resolved on SDS-PAGE and analyzed by western blotting
for detection of associated proteins. Equal protein loading was
confirmed using anti-histone H1 antibody.
Reverse transcriptase-PCR. Total RNA (2 µg) each from
untreated, placebo-/sulphur-treated NSCLC cells was
extracted by Trizol (Invitrogen) and was reverse transcribed
and then subjected to PCR with enzymes and reagents of the
RTplusPCR System (Eppendorf, Hamburg, Germany) using
GeneAmpPCR 2720 (Applied Biosystems, Foster City, CA,
USA) (40). The cDNAs were amplified with primers specific
for Bax (5'-GGAATTCCAAGAAGCTGAGCGAGTGT-3'/
5'‑GGAATTCTTCTTCCAGATGGTGAGCGAG-3'), Bcl-2
(5'-CCTGTGCCACCATGTGTCCATC-3'/5'-GCTGAGAACA
GGGTCTTCAGAGAC-3') and GAPDH (internal control:
5'-TGATGACATCAAGAAGGTGGTGAAG-3'/5'-TCCTTGG
AGGCCATGTAGGCCAT-3').
Chromatin immunoprecipitation (ChIP). ChIP assays were
carried out for identification of p53 and p65NFκ B binding
region on Bax and Bcl-2-promoters respectively, using a
ChIP assay kit (Millipore) according to the manufacturer's
575
instructions. PCR assay was performed using primer sets as
follows: Bcl-2 forward primer 5'-GATTCCTGCGGATTGACA
TTTC-3', Bcl-2 reverse primer 5'-CATCAATCTTCAGCACTC
TCC-3'; Bax forward primer 5'-TCAGCACAGATTAGTTT
CTG-3', Bax reverse primer 5'-GGGATTACAGGCATGAG
CTA-3'. Extracted DNA (2 µl) was used for 45 cycles of
amplification in 5 µl of reaction mixture under the following
conditions: 95˚C for 30  sec, 56˚C for 30  sec and 72˚C for
60 sec. The PCR products were analysed by 2% agarose gel
electrophoresis (41,42).
Statistical analysis. Values are shown as standard error of
mean, except when otherwise indicated. Data were analyzed
and, significance (P<0.05) of the differences between mean
values was determined by a Student's t-test.
Results
Sulphur induces non-small cell carcinoma apoptosis. The
effect of sulphur, a homeopathic drug, on the viability of
human NSCLC cell line (A549) and normal peripheral blood
mononuclear cell (PBMC) was examined at different poten-
cies of sulphur, i.e., 6C, 30C, 200C, where for each potency
a differential dose of 0-30 µl/ml was applied (Fig. 1A and B).
The percent cell death was scored by trypan-blue dye-exclusion
assay. It was observed that among all the potencies of sulphur,
30C potency resulted in most significant decrease (p<0.001) in
cell viability (Fig. 1A). Moreover, at 20 µl/ml dose of 30C, the
percent A549 cell death reached its optimum (Fig. 1A) while
under the same conditions the PBMC viability was found to
be >90% (Fig. 1B). These results indicating the better efficacy
of 20 µl/ml dose of 30C sulphur in A549 cell killing with
minimum toxicity led us to perform all further experiments
using this particular potency and dose of sulphur. The time-
dependent effect of sulphur 30C (20 µl/ml), in comparison to
placebo, on A549 and PBMC cells was examined at different
time intervals (0-48 h) and percentage cell death was assessed.
Sulphur 30C, at a concentration of 20 µl/ml, exerted time-
dependent significant death in A549 cells (Fig. 1C). However,
significant cell death in PBMCs was noted from 24 h onwards
following the treatment (Fig. 1D).
Next, to confirm the nature of cell death as apoptosis,
we utilized double labelling techniques using Annexin
V-FITC/7-AAD to distinguish between apoptotic and
necrotic cells. Our flow cytometric data demonstrated that
in comparison to placebo-treated A549 cells, sulphur-treated
unfixed A549 cells showed Annexin V-FITC binding with
minimum 7-AAD binding (Fig. 1E) indicating that the mode
of cell death was apoptosis but not necrosis. These findings
were re-confirmed by the development of nuclear blebbing
as evidenced by DAPI-stained fluorescent images of sulphur-
treated A549 cells (Fig. 1F). These data together supported
the notion that sulphur 30C asserts apoptogenic effect in the
NSCLC A549 cells.
Sulphur decreases the survival advantage of NSCLC cells by
perturbing nuclear translocation of p65NFκB. Since p65NFκ B
has been reported to be globally involved in survival of cancer
cells (27,28,43), we examined whether sulphur suppresses this
pathway to combat NFκ B-mediated survival to induce apop-
576 Saha et al: Sulphur induces apoptosis in non-small cell carcinoma cells

Figure 1. Sulphur induces tumor apoptosis in NSCLC cells. (A) The number of viable A549 cells following exposure to different potencies (6C, 30C and 200C)
of placebo and sulphur at different concentrations (10, 15, 20 and 30 µl/ml) was determined by trypan-blue dye exclusion assay, and the data are represented
graphically (*P<0.05, ***P<0.001 when compared with the respective placebo-treated group). (B) Graphical representation showing percentage of cell death of
PBMC confirms that sulphur does not induce apoptosis in normal cells (P<0.001 when compared between survival percentages of un-/sulphur-treated PBMCs).
The percent cell death was scored by trypan-blue dye-exclusion assay. (C) Time-dependent effect of sulphur 30C (20 µl/ml), in comparison to placebo, on A549
examined at different time intervals (0-48 h). (D) Time-dependent effect of sulphur 30C (20 µl/ml), in comparison to placebo, on PBMC was examined (*P<0.05
and **P<0.001 when compared with respective control/treated groups). (E) The nature of sulphur induced A549 cells was assayed flow cytometrically using
Annexin V-FITC/7-AAD double labelling assay. (F) DAPI staining revealed nuclear morphology of apoptotic cells (blebbing and fragmentation) as indicated
by arrowheads in sulphur treated sample when visualized under a fluorescence microscope. Bar length in images indicate 20 µm. Values are the mean ± SEM
of three independent experiments in each case or representative of a typical experiment.

tosis in drug-resistant NSCLC cells. Our search revealed that
sulphur treatment efficiently blocked nuclear translocation of
NFκ B in NSCLC cells as observed by both western blotting
(Fig. 2A) and confocal imaging experiments (Fig. 2B). In
addition, the mRNA and protein levels of NFκ B-target gene,
Bcl-2, was found to be downregulated by sulphur treatment in
NSCLC cells (Fig. 2C). We further observed that transfecting
NSCLC cells with super repressor Iκ Bα-SR-cDNA decreased
Bcl-2 followed by significant apoptosis in response to sulphur
(Figs. 2D and E). On the other hand NSCLC cells expressing
p65NFκ B-cDNA manifested enhanced Bcl-2 with significant
resistance upon sulphur exposure (Fig. 2D and E). The anti-
apoptotic role of NFκ B-dependent Bcl-2 upregulation was
re-confirmed by evaluating response of Bcl-2-engineered cells
towards sulphur treatment. Overexpression of Bcl-2 in NSCLC
cells bestowed them with survival advantage upon sulphur
treatment, whereas transfection with Bcl-2-siRNA efficiently
enhanced sulphur-induced apoptosis (Fig. 2F). Collectively,
these results confirmed that p65NFκ B activation and subse-
quent Bcl-2 upregulation were primarily involved in survival
of NSCLC cells which upon inhibition by sulphur induced
apoptosis in these cells.
Sulphur treatment triggers the p53-mediated mitochondria-
dependent apoptotic pathway in NSCLC cells. Since
inhibition of p65NFκ B activity in NSCLC cells induced a
powerful apoptotic response, we predicted the involvement
of the cellular apoptotic proteins during sulphur-induced
apoptosis. We evaluated the status of apoptotic proteases,
i.e., caspase‑9 and caspase-3, respectively, in response to
sulphur treatment. It was noted that sulphur treatment signifi-
cantly upregulated levels of cleaved caspase‑9 and caspase-3
in A549 cells (Fig. 3A). Since tumor suppressor protein p53
plays an important role in canonical apoptotic pathway, the
above results tempted us to compare the p53 activation status
upon sulphur exposure in NSCLC cells. Results of Fig. 3B
left panel revealed that sulphur induced p53 expression in
A549 cells when compared to untreated cells. These results
 INTERNATIONAL JOURNAL OF ONCOLOGY 47: 573-582, 2015 577

Figure 2. Sulphur decreases the survival signal of NSCLC cells by restraining nuclear translocation of NFκ B. (A) Expression of nuclear (N) and cytosolic (C)
p65NFκ B was determined by western blot analysis in the A549 cells treated with placebo and sulphur at 30C (20 µl/ml). (B) Nuclear and cytosolic expression
of p65 was visualized by confocal microscopy after fixation and permeabilization of control, placebo and sulphur treated A549 cells using the anti-p65NFκ B
antibody. (C) Untreated-/placebo-/sulphur-treated A549 cells were subjected to RT-PCR/western blot analysis to determine the expression profile of Bcl-2 at
mRNA and protein levels. (D) Protein expression of Bcl-2 was determined by western blot analysis in Iκ Bα-SR-cDNA or p65NFκ B-cDNA transfected A549
cells in the presence or absence of placebo or sulphur. (E) In a parallel experiment, the percentage of apoptosis was determined by Annexin V-FITC/7AAD
staining, and data are presented graphically (***P<0.001 when compared with the sulphur-treated group). The efficiency of Iκ Bα-SR-cDNA and p65NFκ B-
cDNA transfection was also verified by western blot analysis (inset). (F) Presentation of the percentage of apoptosis in Bcl-2-cDNA or Bcl-2-siRNA transfected
A549 cells in the presence or absence of placebo or sulphur, as determined by Annexin V-FITC/7AAD staining (***P<0.001 when compared with the sulphur-
treated group). The efficiency of Bcl-2-cDNA and Bcl-2-siRNA transfection was also verified by western blot analysis (inset). α-actin was used as an internal
loading control. Values are the mean ± SEM of three independent experiments in each case or representative of a typical experiment.

were further confirmed by confocal microscopy, the results
of which not only demonstrated accumulation of p53 protein
but also its translocation from cytosol to nucleus of sulphur-
exposed A549 cells (Fig. 3B, middle panel). Furthermore,
findings of western blot analysis verified greater transloca-
tion of p53 from cytosol to nucleus upon sulphur exposure
in A549 cells (Fig. 3B right panel). It is acknowledged that
p53 when localized in nucleus transactivates its downstream
target genes (31). We, therefore, next assessed the status of its
trans-activated gene product, i.e., Bax, by western blot and
RT-PCR analyses in sulphur-treated and untreated A549 cells.
Our results re-established that in comparison to un-/placebo-
treated tumor cells, sulphur-exposed cells showed increase in
the levels of p53 trans-activated gene product Bax (Fig. 3C)
Downstream of Bax, increase in cytosolic cytochrome c with
its concomitant decrease in mitochondria (Fig. 3D) were
observed and also significant mitochondrial transmembrane
potential (MTP) loss is observed in sulphur-exposed A549
cells as compared to control and placebo-treated A549 cells
(Fig. 3E).
Next, A549 cells were transfected with Bax-siRNA or
pre-treated with cyclosporine A (CsA), mitochondrial pore
578 Saha et al: Sulphur induces apoptosis in non-small cell carcinoma cells

Figure 3. Sulphur treatment elicits the p53-mediated mitochondrial apoptotic pathway in A549 cells. (A) Expression profile of active caspase-3 and active
caspase‑9 in un-/placebo-/sulphur-treated A549 cells was observed by western blot analysis. (B) Expression of total p53 in un-/placebo-/sulphur-treated A549
cells was determined by western blot analysis (left pannel) and confocal microscopy (middle panel). Cytosolic (C) and nuclear (N) expression of p53 was
observed by western blotting in un-/placebo-/sulphur-treated A549 cells (right pannel). (C) Untreated-/placebo-/sulphur-treated A549 cells were subjected to
RT-PCR/western blot analysis to determine the expression profile of Bax at mRNA and protein levels. (D) Mitochondrial and cytosolic expression of Bax
and cytochrome c was determined by western blotting. (E) Presentation of mean DiOC6 fluorescence to determine mitochondrial transmembrane potential
of untreated-/placebo-/sulphur-treated A549 cells as determined by flow cytometry. (F) Expression of Bax, active caspase‑9 and caspase-3 was observed by
western blotting in control A549 cells and A549 cells transfected with Bax-siRNA or pre-treated with cyclosporine A (CsA), prior to sulphur treatment. In a
parallel experiment, the percentage of apoptosis was determined by Annexin V-FITC/7AAD staining, and data are presented graphically (***P<0.001 when
compared with the sulphur-treated group). α-actin and MnSOD was used as an internal loading control. Values are the mean ± SEM of three independent
experiments in each case or representative of typical experiment.

formation blocker, prior to sulphur treatment for validation
of the involvement of Bax in mitochondria cascade-mediated
NSCLC apoptosis. Silencing Bax, or CsA pre-treatment
significantly decreased percent apoptosis of NSCLC cells and
also down-modulated the expression levels of caspase‑9 and
caspase-3 (Fig. 3F). Taken together these findings validated
the contribution of Bax in sulphur-induced A549 cell apop-
tosis via mitochondrial death cascade.
Inhibition of p65NFκ B by sulphur triggers p53-mediated
apoptosis in NSCLC cells. Next, we verified whether sulphur-
induced cancer cell death is p53-dependent or not. To this
end, human cancer cell lines with differential p53 status,
e.g., wild-type p53-expressing and p53-shRNA-transfected
A549 cells were tested for sulphur-dependent apoptosis by
scoring the number of Annexin V-positive cells flow cyto-
metrically (Fig. 4A). Interestingly, sulphur 30C at 20 µl/ml
dose significantly (p<0.001) induced apoptosis in wild-type
p53-expressing A549 cells. The apoptogenic insult asserted by
sulphur after minimization of placebo effect in A549 cells were
31%, while p53-knockdown cells resisted such insult (Fig. 4A).
These results indicated the contribution of functional p53 in
sulphur-induced cancer cell apoptosis.
In parallel experiment when A549 cells were transfected
with p53-cDNA, p53 expression though increased, Bax
expression level failed to reach that of sulphur-treated A549
cells (Fig. 4B). These findings revealed that increasing p53
levels alone in NSCLC cells failed to restore p53 transcrip-
tional functions and to induce apoptosis (Fig. 4C). This
raised the possibility of the involvement of p53 transcrip-
tional ‘inhibitor(s)’ in NSCLC cells that somehow opposed
p53-dependent transcription of apoptotic genes. Sulphur, on
the other hand, by restraining this inhibitor might have acti-
vated the p53-transcriptional program. Since our previous
results (Fig. 2A and B) have demonstrated sulphur-induced
inhibition of NFκ B activation, we hypothesized that activated
NFκ B might be blocking p53-dependent apoptotic program
in NSCLC cells. To confirm this hypothesis we utilized
Iκ Bα-SR-cDNA transfected cells and checked p53-dependent
execution of apoptosis in these cells upon transfection with
p53-cDNA. Indeed these transfectants displayed robust p53
induction along with upregulation of Bax (Fig. 4B). Activation
 INTERNATIONAL JOURNAL OF ONCOLOGY 47: 573-582, 2015 579

Figure 4. Sulphur ensures p53-mediated apoptosis in NSCLC cells by inhibiting p65NFκ B. (A) The percentage of apoptosis was determined in control or p53-
shRNA transfected A549 cells in presence or absence of placebo or sulphur by Annexin V-FITC/7AAD staining, and data are presented graphically (***P<0.001
when compared with the sulphur-treated group). The efficiency of p53-shRNA transfection was also verified by western blot analysis (inset). (B) Protein
expression of Bax and active caspase-3 was determined by western blot analysis in Iκ Bα-SR-cDNA or p53-cDNA transfected A549 cells in the presence or
absence of placebo or sulphur at 30C (20 µl/ml). (C) In a parallel experiment, the percentage of apoptosis was determined by Annexin V-FITC/7AAD staining,
and data are presented graphically (***P<0.001 when compared with the sulphur-treated group). The efficiency of p53-cDNA transfection was also verified by
western blot analysis (inset). α-actin was used as an internal loading control. Values are the mean ± SEM of three independent experiments in each case or
representative of a typical experiment.

of caspase-3 in these cells (Fig. 4B) finally confirmed that
NFκ B intervened the functioning of p53-dependent apoptotic
program.
All these results together signified that sulphur by inhib-
iting p65NF κ B-governed survival signalling skewed the
cellular microenvironment in favor of p53-transcriptional
activation to result in apoptosis.
Sulphur rescues p300 from p65NFκB to establish p53-p300
collaboration in NSCLC cells. We next attempted to unveil
the detail mechanisms underlying NFκ B-mediated inhibition
of p53 transcription functions. Recent studies indicate that
the transcriptional activity of p53 is regulated by its interac-
tion with the transcriptional co-activator p300 (27,31). To
verify the effects of sulphur on p53-p300 cross-talk, if any,
we immunoprecipitated nuclear p300 in NSCLC cells treated
with placebo, or sulphur and verified its interaction with p53
by western blotting. It was observed that in contrast to placebo,
sulphur induced p53-p300 interaction (Fig. 5A). Consistently,
it was further observed by ChIP analysis that sulphur treat-
ment in NSCLC cells enhanced p53 binding on Bax promoter,
which is a pre-requisite for transcriptional activation of Bax
(Fig. 5B). This subsequently enabled p53-dependent apop-
tosis as observed earlier (Fig. 3A). Since, sulphur triggered
p53-p300 interaction in cells where p65NFκ B activation
was not taking place, we proposed that nuclear translocation
of p65NFκ B in untreated p53-cDNA transfected A549 cells
might have sequestered p300 thereby abridging p53-p300
cross-talk. As anticipated, these untreated p53-cDNA trans-
fected A549 cells manifested significant p65NFκ B-bound
p300 in their nuclear lysates (Fig. 5C). Furthermore, upon
genetically perturbing nuclear translocation of p65NFκ B in
these p53-cDNA trasfected A549 cells, significant increase in
p53-p300 interaction was observed with concomitant decrease
in p65NFκ B-bound p300 (Fig. 5D). Interestingly sulphur
treatment inhibited p65NF κ B-p300 cross-talk (Fig. 5E)
thereby preventing p65NFκ B binding on Bcl-2 promoter to
initiate p65NFκ B-mediated Bcl-2 transcription (Fig. 5F).
These results indicate a competition between NFκ B and p53
for availing p300, and depending on the relative availability,
the winner, and the fate of the cells are decided.
In summary, the above results conclude that in drug-
resistant NSCLC cells, p65NFκ B competes with p53 for
the transcriptional co-activator p300 thereby inhibiting the
apoptotic program and upregulates the survival-machinery of
the cell. On the other hand, by inhibiting p65NFκ B, sulphur
censored the survival pathway thereby making p300 available
for p53 interaction to ensure the transcription of pro-apoptotic
protein Bax for effective induction of apoptosis in otherwise
resistant NSCLC cells (Fig. 6).
580 Saha et al: Sulphur induces apoptosis in non-small cell carcinoma cells

Figure 5. p53-p300 collaboration is established by sulphur, which rescues p300 from p65NFκ B in NSCLC cells. (A) p53-associated p300 was immunopurified
with anti-p53 antibody from nuclear lysates of un-/placebo-/sulphur-treated A549 cells and were western-blotted (WB) with p300 and p53 antibodies. (B) A
portion of cells from the same experimental set were subjected to ChIP assay for the determination of p53 binding on Bax promoter. (C) p53-/p65NFκ B-
associated p300 was immunopurified with anti-p53/p65NFκ B antibodies from nuclear lysates of control and p53-cDNA transfected A549 cells and were
western-blotted (WB) with p300 antibody. (D) p53-/p65NFκ B-associated p300 was immunopurified with anti-p53 and p65NFκ B antibodies from nuclear
lysates of control, p53-cDNA transfected or Iκ Bα-SR-cDNA transfected A549 cells and were western-blotted (WB) with anti-p300, p53 and p65NFκ B
antibodies (E) p65NFκ B-associated p300 was immunopurified with p65NFκ B antibody from nuclear lysates of un-/placebo-/sulphur-treated A549 cells and
were western-blotted (WB) with p300 and p65NFκ B antibodies. (F) A portion of cells from the same experimental set were subjected to ChIP assay for the
determination of p65NFκ B binding on Bcl-2 promoter. To verify comparable protein input during immunoprecipitation, 20% of supernatant from the nuclear
lysates was blotted with histone H1 antibody.

Discussion

Sulphur has been widely accredited for its antitumor potential

Figure 6. Schematic illustration depicting differential regulation of anti- and
pro-apoptotic networks by sulphur in non-small cell carcinoma cells.
(15-18). Although few reports have also verified the anticancer
effect of this remedy (17,19), detailed report elucidating the
molecular mechanisms underlying the anticancer effect of
sulphur is still warranted. The present study demonstrated that
the antitumor effect of sulphur on non-small cell lung carci-
noma cells was not a ‘placebo effect’ as placebo-(potentized
hydro-alcoholic solution) treated cells failed to show signifi-
cant death when compared to control cells. This study further
revealed that sulphur asserted its effects by re-orienting
the molecular choreography of cancer cells. Importantly,
preferential induction of cytotoxic effects in drug-resistant
NSCLC cells, as compared to normal cells, raised the exciting
possibility for a window of safe and non-toxic therapeutic
opportunity.
Here we report that sulphur induces apoptosis in NSCLC
cells by inhibiting p65NFκ B-mediated survival pathway and
activating p53-apoptotic signaling. In fact, inactivation of
 INTERNATIONAL JOURNAL OF ONCOLOGY 47: 573-582, 2015
NFκ B pathway by sulphur rescued p300 from p65NFκ B and
launched p53-p300 collaboration to induce p53-dependent
Bax-transactivation and instigation of downstream mitochon-
dria-dependent death cascade in NSCLC cells. The regulatory
contribution of NFκ B and p53 to cancer development and
progression is well documented where inactivation of p53
and hyper-activation of NFκ B are the common occurrences
(25-27,44). In agreement with such complex regulation of
NFκ B and p53 at several steps, these transcription factors can
functionally antagonize, cooperate or exhibit independence
(45-48). Likewise in our study we observed that p65NFκ B,
being activated in NSCLC cells, interfered with p53 functions
by p300 sequestration. Inhibition of p65NFκ B by sulphur or
Iκ Bα super repressor rescued p300 from p65NFκ B-clutch to
restrain the resistance pathway. Consistently there are studies
reporting that p65NFκ B has a high-affinity for p300 that
may lead to its sequestration thereby making it unavailable to
other transcription factors (35). In line with these studies we
observed that upon sulphur treatment, inhibition of p65NFκ B
rescued p300 making it available to other transcription factor/s
like p53 in the present case, thereby allowing p53-dependent
transactivation of apoptotic proteins.
Our findings were consistent with those of Webster and
Perkins (48) who first reported that the RelA (p65) subunit of
NFκ B antagonized p53 transactivation through sequestration
of the p300 and CBP co-activators. It is acknowledged that
p300 and CBP participate at various stages of the p53 response,
functioning as essential co-activators in p53-dependent trans-
activation of target genes (49). They promote transcription
of specific p53 targets by two mechanisms. First, p300 and
CBP are recruited by p53 to target gene promoters where they
acetylate histones. Secondly, p53 acetylation, secondary to
DNA damage, stabilizes the p53-DNA complex at target gene
promoters (49). Similarly acetylation of p65NFκ B is impor-
tant for p65NFκ B-DNA binding activity and p300 activation
is known to enhance p65NFκ B acetylation (50). The N- and
C-terminal domains of both CBP/p300 functionally interact
with a region of p65NFκB containing the transcriptional activa-
tion domain and thereby promote the trans-activating functions
of p65NFκ B transcription factors (50). Therefore, our results
along with others, suggest that p65NFκB and p53 compete for
transcriptional co-activator p300 and depending upon whether
p65NFκB or p53 hires p300, execution of downstream effector
pathways oscillates between survival and apoptotic responses
(Fig. 6).
In conclusion, our study for the first time indicated an
apoptosis-inducing capability of sulphur in otherwise drug-
resistant NSCLC cells by shifting the cellular milieu from
NFκ B-mediated survival environment towards p53-mediated
apoptosis. Even though further investigations and clinical
trials are needed, overall these findings provide evidence for
a molecular signature of the apoptotic effects of sulphur on
NSCLC cells.
Acknowledgements
The authors are thankful to Mr. Uttam Ghosh and Mr. Ranjan
Dutta for their technical help. This study was supported by
the grants from Central Council for Research in Homeopathy
(CCRH), Government of India.
581
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